1. Field of the Invention
Genetic manipulation of plant cells is an area of widespread interest. The ability to confer desirable traits, such as disease resistance, environmental resistance, enhanced yield, and the like, is of great economic and scientific importance. In order to genetically manipulate plant cells, it is necessary to develop systems for the delivery and integration of desired structural genes into the plant genome.
Heretofore, research has centered on the use of DNA plant viruses and the Ti plasmid of Agrobacterium tumefaciens for use as vectors to introduce DNA into higher plants. Both of these proposed systems suffer from inherent limitations. DNA plant viruses, such as the cauliflower mosaic virus (CaMV), have restricted host ranges and often lose infectivity after manipulation. Moreover, recombinant CaMV molecules might be too big to be packaged into virus particles, limiting their usefulness. Also, there is no evidence that CaMV DNA integrates into host cell chromosomes. Without integration, it is unlikely that the recombinant molecule would be transmitted to progeny of the infected plant.
In contrast, incorporation of Ti plasmid DNA into plant cells has been reported. Hernalstens et al. (1980) Nature 287:654-656, and Garfinkel et al. (1981) Cell 27:143-153. More importantly, it has been found that foreign DNA inserted into the Ti plasmid in the region near the T-DNA can be incorporated into the plant genome. Despite these promising results, use of the Ti plasmid as a vehicle for introducing DNA into higher plants remains problematic. The Ti plasmid can be maintained only in A. tumefaciens which is not a convenient host for in vitro manipulation. Also, the Ti plasmid is very large (usually above 200 kbp) further rendering manipulation difficult, particularly when relatively large DNA fragments have been inserted. Finally, maintenance of the Ti plasmid in A. tumefaciens is less stable than would be desired.
For these reasons, it would be desirable to provide a cloning vehicle which could be stably maintained in other more convenient hosts, such as E. coli; as well as A. tumefaciens. Such vectors would allow insertion and manipulation of foreign DNA in the E. coli followed by introduction into A. tumefaciens. It is further desirable that the vectors include a plurality of selectable markers and be sufficiently small in size to accommodate relatively large DNA inserts. Finally, it would be desirable that the vector be able to transfer the inserted foreign DNA into the genome of a higher plant.
2. Description of the Prior Art
The pTAR plasmid was described by Kado et al. (1981) J. Bacteriol. 145:1365-1373. Plasmid pUCD400 (formerly pCK2D) was first described in Kado et al. (1982) Proc. Fourth Intl. Symp. Genetics Indust. Microorg., Kyoto, Japan.